The porcine islet-derived organoid showed the characteristics as pancreatic duct

Organoid is a tissue-engineered organ-like structure that resemble as an organ. Porcine islet-derived organoid might be used as an alternative donor of porcine islet xenotransplantation, a promising therapy for severe diabetes. In this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets as a cell source for transplantation. Isolated porcine islets were 3D-cultured using growth factor-reduced matrigel in organoid culture medium consist of advanced DMEM/F12 with Wnt-3A, R-spondin, EGF, Noggin, IGF-1, bFGF, nicotinamide, B27, and some small molecules. Morphological and functional characteristics of islet organoids were evaluated in comparison with 2D-cultured islets in advanced DMEM/F12 medium. Relatively short-term (approximately 14 days)—cultured porcine islet organoids were enlarged and proliferated, but had an attenuated insulin-releasing function. Long-term (over a month)—cultured islet organoids could be passaged and cryopreserved. However, they showed pancreatic duct characteristics, including cystic induction, strong expression of Sox9, loss of PDX1 expression, and no insulin-releasing function. These findings were seen in long-term-cultured porcine islets. In conclusion, our porcine islet organoids showed the characteristics of pancreatic ducts. Further study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Next, we evaluated gene expression among the three groups.In terms of genes correlated to carbohydrate antigens, expression of Ggta1p and Cmah in islet organoids was significantly enhanced compared with that in overnight-and 11 day-cultured islets (Fig. 3A and B).In terms of pancreatic differentiation, we assessed the expressions of Pdx1, Ptf1a, Sox9 and Neurog3.Pdx1 is expressed on multipotent progenitor cells in pancreatic bud.It is required in the earliest step of pancreatic formation.The expression is maintained till the differentiation into β-cells 19 .PTF1 was firstly identified as an acinar enzyme gene activator 20 .As same as Pdx1, Ptf1a is also expressed in early pancreatic multipotent progenitor cells.After that, Ptf1a-expressed multipotent progenitor cells are differentiated into acinar cells, while Ptf1a null cells are into bipotent progenitor cells which enable to differentiate into duct/endocrine cells 20 .Sox9 is also expressed on early pancreatic multipotent progenitor cells 21 .As opposed to Ptf1a, the expression of Sox9 is maintained in bipotent progenitor cells and attenuated in acinar cells.Finally, the expression is seen in ductal cells, while disappeared in endocrine progenitors 20 .SOX9 is known as a master regulator of pancreatic differentiation 22 .It acts as a critical maintenance factor of pancreatic progenitors, and contributes to maintenance of pancreatic ducts 23 .Neurogenin 3 work for initiation of the endocrine development.The expression of Neurog3 is seen on endocrine progenitor 20 .In this study, the expression of Pdx1 was attenuated in 11 day-cultured islets.However, its expression in islet organoids was recovered to the same level as that in overnight-cultured islets (Fig. 3C).Expression of Neurog3 tended to be attenuated in 11 day-cultured islets and islet organoids (Fig. 3D).On the other hand, prominent elevation in the expression of Sox9 was seen in islet organoids (Fig. 3E).Regarding Ptf1a, the expression was hardly detected (Ct > 40) in overnight-cultured islets, 11 day-cultured islets and 11 day-cultured islet organoid (data not shown).In terms of islet hormones, attenuation of Ins and Sst expression and elevation of Gcg expression were seen in islet organoids (Fig. 3F-H).

Long-term cultured islet organoids show the characteristics of pancreatic ducts
Figure 4A shows the scheme to form islet organoids from dispersed islet cells.Dispersed cells were embedded in growth factor-reduced matrigel and cultured in organoid culture medium (Fig. 4B).This followed an original procedure to form organoids using a biopsy sample from the organ.We assessed the characteristics of islet organoids under long-term culture for > 2 months.Porcine islets were obtained from a transgenic pig whose Pdx1 gene promotor was conjugated to the Venus gene encoding a green fluorescent protein (Pdx1-Venus Tg pig) 24 .The information of Pdx1-Venus Tg pig is shown in Table 1.The quality of isolated islets was indicated by 95% cellular purity (Fig. 4C), 93.5 ± 2.0% viability (n = 6 islets, Fig. 4D), and GSIS in accordance with the glucose concentration [low glucose: 0.033 ± 0.005 ng/h/IEQ; high glucose: 0.111 ± 0.021 ng/h/IEQ; stimulation index (ratio of insulin secretion between high and low glucose stimulations): 3.42 ± 0.55; Fig. 4E].
Most dispersed islet cells on day 1 were positive for PDX1 with expression of Venus (Fig. 5A).A few spheroids were seen on day 5.They were positive for PDX1, and some of them included cystic components (Fig. 5B).PDX1positive spheroids/cells decreased gradually over time, and few positive cells were found on day 14 (Fig. 5C).On day 23, most cells had changed to a cystic construction.The morphological characteristics of islet organoids were similar to those of pancreatic ducts.Some PDX1-positive cells were detected in the cystic construction (Fig. 5D).The cystic components were enlarged and proliferated in the organoid culture (Fig. 5E).On day 45, PDX1-positive cells had completely disappeared (Fig. 5F).
Figure 6 shows the gene expression of islet organoids derived from the Pdx1-Venus Tg pig on day 45 compared with overnight culture.In terms of genes correlated to carbohydrate antigens, significant elevations were seen in Ggta1p and Cmah expression in islet organoids, as same as islet organoids on day 11 (both p < 0.0001; Fig. 6A and  B).In terms of pancreatic differentiation and endocrine hormones, expression of Pdx1 was significantly decreased and expression of Ins, Gcg, and Sst had completely disappeared on day 45 (all p < 0.05; Fig. 6C-F).Recovery of Pdx1 expression and elevation of Gcg expression on day 11 were cancelled on day 45.However, elevation of Sox9 expression was also seen on the day (p < 0.01; Fig. 6G).

Transplanted islet organoids show the characteristics of pancreatic ducts
We performed xenotransplantation of porcine islet organoids into diabetic nude mice.Prior to transplantation, the characteristics of the islet organoids were assessed histologically.Figure 8A shows a histological image of islet organoids on day 52.They consisted of single and multiple layered columnar cells.Some cells formed rosette shapes.The shapes of nuclei were round and oval.Many of them included nucleoli.All cells comprising islet organoids were positive for CK19.Some of them were positive for SLA1 on the cell membrane (Fig. 8B).However, few cells in the tissues were positive for C-peptide and its expression was mostly weak (Fig. 8C). Figure 9 shows the therapeutic effects of islet organoids derived from microminipigs and cultured for 27 days (Fig. 9A).One hundred islet organoids were xenotransplanted into the renal subcapsular space in diabetic nude mice (Fig. 9B).
They were engrafted on POD 28 with feeding blood supplies from recipients (Fig. 9C).No recovery of blood glucose or elevation of plasma porcine insulin was observed after xenotransplantation.A slight increase in blood glucose and decrease in porcine insulin were seen after graftectomy (Fig. 9D and E).Histological assessment revealed that the engrafted islet organoids formed ductal structures.They were positive for CK19 and some were double positive with SLA1.Most of them were negative for porcine C-peptide, and few positive cells were seen (Fig. 9F).These outcomes of islet organoid xenotransplantation coincide the characteristics of these islet organoids, including GSIS, expression of genes correlated with pancreatic differentiation and endocrine hormones, which showed the attenuation of functions as islets.

Discussion
An organoid is a self-organized 3D tissue derived from pluripotent or tissue stem cells that imitates important functional, structural, and biological characteristics of the original organ 1, 25 .Huch and colleagues developed the methodology for pancreatic organoid formation.The organoids were derived from murine pancreatic duct tissue.They found that pancreatic duct tissue cultured in matrigel grew and acquired cystic components with buddings.
The pancreatic organoids harbored the characteristics of both pancreatic ducts, including SOX9 expression, and  www.nature.com/scientificreports/endocrine tissue, including some insulin-positive cells 26 .Subsequently, Azzarelli and colleagues enhanced the β cell characteristics in pancreatic organoids derived from rodent pancreatic ducts by induction of neurogenin 3 27 .Kim et al. developed pluripotent stem cell-derived islet organoids that produced endocrine hormones by induction of differentiation into β cells and the formation of spheroids 28 .Additionally, Wang et al. developed islet organoids that could be passaged and retained the endocrine function long term by co-culturing islet cells with endothelial cells 8 .
In this study, we clarified the characteristics of our porcine islet organoids.Porcine islet-derived organoids that improve the endocrine function of diabetic patients after transplantation might contribute to the promotion of porcine islet xenotransplantation and curing severe diabetes.We assessed the characteristics of relatively short-term-cultured porcine islet organoids (11 days).The organoid culture medium contained Wnt, R-spondin, and Noggin.R-spondin is an enhancer of Wnt/β-catenin that promotes cellular proliferation and maintains the differentiation behavior of stem cells 29 .Noggin is an extracellular antagonist of bone morphogenetic protein and plays an important role in the formation of neural, intestinal, hepatic, and pulmonary organoids 30 .These factors are required to maintain the condition of stem cells in porcine islets.On day 11, the porcine islets had enlarged compared with the original porcine islets, indicating that cell proliferation was promoted by the organoid culture, particularly the supplements.Islets are a well-differentiated tissue with poor cellular proliferation.Enhanced cell proliferation is considered to be a preferable characteristic for stocking because cell proliferation after cryopreservation recovers the condition of organoids injured by cryopreservation.However, islet organoids, including cystic/ductal structures, had an attenuated endocrine function.Our islet organoids secreted insulin under glucose stimulation.However, this function was significantly inferior to that of islets on day 11.Conversely, glucagon secretion of islet organoids was significantly higher than islets on the day.Recovery of Pdx1, elevation of Sox9 and Gcg expression, and attenuation of Ins and Sst expression were seen in the islet organoids.These results indicated that relatively short-term-cultured islet organoids showed the characteristics of pancreatic ducts, but Next, we implemented long-term (45 days) organoid culture of Pdx1-Venus Tg porcine islets.This model was clearly suitable to follow PDX1-positive tissue considered to be islets.Most islet cells were positive at 1 week, and cystic and ductal structures were seen during this time.On day 14, PDX1-positive tissue was significantly decreased.On days 23 and 31, islet organoids with a cystic structure were enlarged and proliferated with attenuation of PDX1 expression.On day 45, PDX1-positive tissue had completely disappeared.The tendency of gene expression in islet organoids was similar to that on day 11.However, both recovery of Pdx1 expression and elevation of Gcg expression seen on day 11 were completely cancelled on day 45.Sox9 expression was also seen in the islet organoids.Furthermore, expression of carbohydrate antigen-correlated genes (Ggta1p and Cmah) was strongly elevated in islet organoids.Thus, long-term-cultured islet organoids harbored similar characteristics to pancreatic ducts and the characteristics of endocrine tissue had completely disappeared.The organoid culture medium contained B27 and nicotinamide, which contribute to β cell proliferation 31,32 , but attenuation could not be prevented in this study.It was also revealed that the islet organoids could be cryopreserved in this study, but the characteristics were likely those of pancreatic ducts.We considered that these consequential events of porcine islets seen under organoid culture resembled those of long-term-cultured porcine islets (Fig. 10).Our previous study revealed that porcine islets cultured for 28 days acquire cellular proliferation and partial recovery of the endocrine function at 37 °C.The long-term culture stressed porcine islets, but they resisted the stress by incubation at 37 °C.We considered that islet stem cells, including pancreatic stellate cells, might contribute to islet regeneration under our culture conditions 15 .However, 28 days might be the time limit for islet regeneration.Schmied and colleagues showed that ductal formation begins in cultured hamster islets on day 7, which proliferate gradually.On day 35, undifferentiated ductal cells expressing cytokeratin and antitrypsin, and not expressing insulin-promoting factor 1, NKx6.1, Pax6, or NeuroD correlated islet development 33 .Furthermore, they found that 60 day-cultured human islets changed into undifferentiated ductal cells expressing cytokeratin 7 and 19 34 .These data showed that long-term culture for > 1 month might convert islets into ductal cells.Moreover, organoid culture might promote such conversion.
Our study revealed that used supplements for promoting organoid formation did not contribute to maintain the characteristics as islet.Among the supplements, we used EGF, nicotinamide and B27, which played a role in inducting differentiation into insulin-producing cells 35 .It might be necessary to use further supplements, such as activin A, retinoic acid, for mainraining characteristics as islet 36 .
In summary and conclusion of this study, we elucidated the characteristics of porcine islet organoids derived from porcine islets.Porcine islet organoids proliferate and can be cultured and passaged for a long time and cryopreserved.However, they show the characteristics of pancreatic ductal cells.These changes were seen in long-term-cultured porcine islets.Therefore, the formation of porcine islet organoids imitates the changes in porcine islets under long-term culture.Futher study is necessary for producing porcine islet-derived organoids having characteristics as islets.

Animals
As donor animals, microminipigs (Fuji Micra Inc., Fujinomiya, Japan) and Pdx1-Venus Tg pigs (kindly provided by Prof. Hiroshi Nagashima, Meiji University) were used.Eight to 12-week-old BALB/cAJcl-nu/nu male mice (CLEA Japan Inc., Tokyo, Japan) were also used as recipients for organoid transplantation.Animals were housed under specific pathogen-free conditions with free access to food and water.The care of mice and experimental procedures complied with the ''Principles of Laboratory Animal Care'' [Guide for the Care and Use of Laboratory

Pancreas collection
The porcine pancreas was harvested as described in our previous studies 15,37 .In brief, a total pancreatectomy was performed under general anesthesia using isoflurane.After heparinization, the pigs were exsanguinated by incising the vena cava in the thoracic cavity, and Belzer UW ® Cold Storage Solution (https:// amn.astel las.jp/ conte nt/ dam/ jp/ amn/ jp/ ja/ di/ pdf/ blz/ Belzer_ UW_ Cold_ Stora ge_ Solut ion.pdf; Preservation Solutions, Inc. Elkhorn, WI) was infused via the abdominal aorta, while abdominal organs were cooled using crushed ice.After flushing out blood, the total pancreatomy was performed.An 18-24 G intravenous catheter was inserted into the pancreatic duct, and cold preservation solutions (ET-Kyoto solution; Cat# 035-13121-2; Otsuka Pharmaceutical Factory, Inc., Naruto, Japan, and ulinastatin; Cat #3999405A2077; Mochida Pharmaceutical Co., Tokyo, Japan) were infused at 1 mL/g pancreas mass.

Porcine islet isolation
Porcine islet isolation was also conducted as described in our previous studies 15,37 .A collagenase solution containing liberase MTF (0.5 g per 1 vial) and thermolysin (15 mg per 1 vial) (Cat# 05339880001; Roche Custom-Biotech, Penzberg, Germany) was instilled into the disinfected pancreas via the catheter placed in the pancreatic duct.The distended pancreas was placed in a Ricordi chamber.The digestion process was initiated by shaking the chamber to circulate the warmed collagenase solution.After stopping digestion and washing the digested pancreatic tissue with RPMI 1640 medium (Cat# 11875085; Thermo Fisher Scientific, Gibco, Waltham, MA) containing 10% inactivated plasma (fetal bovine serum, qualified, United States, Cat #26140079; Thermo Fisher Scientific, Gibco), the tissues were collected in Belzer UW ® Cold Storage Solution.The purification process was performed using IBM 2991 (COBE 2991; Terumo BCT, Tokyo, Japan) by centrifugation with a continuous density gradient between 1.077 and 1.100 g/cm 3 created using Optiprep (Cat# ST-07820; Veritas Co., Tokyo, Japan).After centrifugation, gradient density solutions containing highly purified islets (≥ 70%) were collected.

Cryopreservation of islet organoids
We attempted to cryopreserve the organoids because islets, especially porcine islets, are difficult to cryopreserve because of their vulnerability and maturity.Organoids were suspended in 1 mL Cellbanker 1 (Zenogen Pharma, Koriyama, Japan) and gradually cooled to − 80 °C using a BICELL (Nihon Freezer Co., Tokyo, Japan) to prevent cellular damage.After overnight cooling, the organoids were cryopreserved in liquid nitrogen.Frozen organoids were thawed by warming at 37 °C.

Glucose-stimulated insulin and glucagon secretions
Glucose-stimulated insulin secretion (GSIS) and glucagon secretion (GSGS) of porcine islets and islet organoids and islets was assessed by treatment with various concentrations of glucose.In brief, 300 islet equivalents (IEQs) and organoids were preincubated with 3.3 mM glucose for 60 min.After preincubation, the islets were stimulated with glucose at 3.3 mM (low glucose) or 16.5 mM (high glucose) for 60 min.Insulin and glucagon in culture supernatants were measured using an LBIS Porcine Insulin enzyme-linked immunosorbent assay (ELISA) Kit (Fujifilm Wako Shibayagi Co., Shibukawa, Japan) and a Glucagon ELISA Kit (Wako), respectively.

Real-time reverse transcription-polymerase chain reaction analysis
RNA was extracted from porcine islet samples using TRIzol (Cat# 15596026; Thermo Fisher Scientific, Invitrogen) and purified using a PureLink ® RNA Mini Kit (Cat# 12183018A; Thermo Fisher Scientific) in accordance with the manufacturers' instructions.RNA concentrations were equalized using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).Reverse transcription was performed using a QuantiTect Reverse Transcription Kit (Cat# 205311; Qiagen K.K., Tokyo, Japan).qRT-PCR analysis was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) and Thunderbird SYBR qPCR Mix (Cat# QPS-101: Toyobo Co., Ltd., Osaka, Japan).The primers used for real-time RT-PCR are shown in Table 2. Primers were designed by Fasmac Co., Ltd.(Atsugi, Japan).Relative quantitation was performed using LightCycler Software Version 4.1.The results were normalized to expression of a reference gene (Actb).Data are presented as the fold difference calculated using the 2 −ΔΔCt method.

Induction of diabetes in recipient mice
Diabetes was induced in recipient mice by intravenous injection of streptozotocin (220 mg/kg body weight; Sigma-Aldrich).Mice with blood glucose levels exceeding 400 mg/dL were used as diabetic recipients.

Organoid transplantation
Recipient mice were anesthetized using isoflurane.A dorsal incision was made through the muscle and peritoneum, and the left kidney was mobilized outside the abdomen.The renal capsule was peeled off from the parenchyma to prepare the renal subcapsular space for transplantation of islet organoids.Porcine islet organoids were placed in the space.After transplantation, the kidney was placed back in the abdomen, and the incision was sutured.The function of transplanted islets was assessed by monitoring blood glucose and plasma insulin concentrations.Plasma concentrations of porcine insulin were measured using an LBIS Porcine Insulin ELISA Kit (Fujifilm Wako Shibayagi Co.).

Figure 8 .
Figure 8. Histological characteristics of islet organoids on day 52.(A-C) Histological images of islet organoids on day 52 after HE staining (A) and immunohistochemical staining of CK19 (green), SLA1 (red) (B), and porcine C-peptide (C).(D) and (E) Histological images of porcine pancreas after immunohistochemical staining of CK19 (green), SLA1 (red) (D), and porcine C-peptide (E).Pancreatic ducts were positive for CK19, and islets were positive for porcine C-peptide.SLA1 was diffusedly stained in the cell membrane of pancreatic cells.Nuclear staining was performed using DAPI (blue).Scale bar = 200 µm.

Figure 10 .
Figure 10.Events in porcine islets correlated to preservation of islets and differentiation into pancreatic ducts seen in long-term culture (upper) and organoid (lower) culture.

Table 2 .
Porcine primers for real-time reverse transcription-polymerase chain reaction analysis.